Endocytosis via clathrin-coated pits is a multipstep process (1, 2). Clathrin heavy and light chains are brought to the plasma membrane through association with a heterotetrameric complex known as clathrin adaptor complex 2, o-r AP-2. At coated pits, the membrane is bent through the assembly of clathrin triskelion into a caged lattice. The GTPase Dynamin is also recruited to the neck of coated pits where it assembles into a collar for vesicle fission (4, 5). Recruitment of Dynamin to coated pits is believed to require the Amphiphysin I/II heterodimer (6), as these proteins bind Dynamin in vivo and amphiphysin proteins are required for endocytosis in yeast (7). In addition, ectopic expression of either Amphiphysin I or II by themselves (6), or the isolated SH3 domain of Amphiphysin I blocks endocytosis (8, 9). Recent data has revealed that the Rab5 small GTPase is required for sequestration of ligands such as transferrin and Epidermal Growth Factor into coated pits in vitro (10). The mechanism by which these components interact to regulate coated pit assembly, cargo sequestration, followed by vesicle fission is not yet understood.
From biochemical, cell biological and genetic analysis it is clear that additional components such as kinases, phosphatases, ubiquitin conjugating enzymes as well as lipid modifying enzymes are required for clathrin-coat and vesicle formation (1, 11, 12, 13, 14, 15, 16, 17, 18, 19). Indeed there is also strong evidence for a requirement of the actin cytoskeleton in endocytosis and several proteins which may facilitate this connection (1, 7, 20, 21, 22, 23, 24, 25).
The Eps15 protein was discovered in a search for substrates of the Epidermal Growth Factor Receptor (26). In 1995, Benmerah et. al. reported that Eps15 is constitutively associated with α-adaptin of the AP2 complex (27). The Eps15 protein has also been localized to the neck of clathrin-coated pits by immunoelectron microscopy (28, 29). Recently, two groups have used dominant inhibitory mutants of Eps15, or antibodies against Eps15 (or the related protein Eps15R), to demonstrate that Eps15 proteins are required for endocytosis via clathrin-coated pits (30, 31). Eps15 contains three large structural domains (26, 32). The N-terminal third contains three copies of an EH domain (for Eps15 Homology domain) (32, 33). The central region of Eps15 forms an extended coiled-coil, which is followed by a complex C-terminus containing SH3-binding motifs (34), a large number of DPF repeats (Aspartic acid-Proline-Phenylalanine), and α-adaptin binding sequences (31, 35, 36). The full length Eps15R protein has a similar overall organization (34). Both Eps15 and Eps15R can be alternatively spliced to produce numerous smaller proteins(37).
A protein with similar overall organization has been identified in Saccharomyces cerevisiae named Pan1p. Genetic analysis of PAN1 has revealed that this gene is required for endocytosis and for organization of the actin cytoskeleton (23, 38). Like Eps15 and Eps15R in mammals, the Pan1p protein has N-terminal EH domains followed by a central coiled-coil domain and C-terminal proline-rich sequences. A second EH domain containing protein, End3p, has also been described in S. cerevisiae which is required for endocytosis and regulation of the actin cytoskeleton (21, 39). Co-immunoprecipitation studies have shown that Pan1p and End3p form a complex in vivo (40). Indeed, overexpression of End3p can suppress the phenotype of pan1–4 hypomorphic mutants, and Pan1p is mislocalized in end3 mutants indicating that these proteins function together (40). Additional studies have revealed that the EH domains of Pan1p bind to yeast homologues of mammalian clathrin-binding proteins, AP180 and CALM (yAP180A and yAP180B), through NPF motifs (Asparagine-Proline-Phenylalanine) in the yAP180 C-termini(25). These data have led to a proposal that the Pan1p:End3p complex functions as a multivalent adaptor to coordinate protein—protein interactions during endocytosis (25, 40). At least two additional proteins are predicted to bind to the Pan1p:End3p complex in vivo, as strong genetic interactions have been detected between PAN1 and SJL1(25), and between PAN1 and RSP5 (41). SJL1 encodes a phosphatidylinositol polyphosphate-5-phosphatase protein which is related to mammalian synaptojanin (42) and has a C-terminal NPF motif predicted to bind to EH domains in Pan1p (or End3p) (25, 43). RSP5 encodes an E3 ubiquitin-protein ligase which may bind to the C-terminal polyproline sequences in Pan1p through one of its three WW domains (25).
Numerous SH3 domain containing proteins have been implicated in the regulation of endocytosis (44). These include Amphiphysin I(45) and II(6, 46, 47, 48), Rsv161/Rsv167(7), Actin Binding Protein-1(49), Endophilin/SH3P4/8/13 (50, 51) and Grb2 (52). Kay and coworkers have reported the isolation of several novel SH3 encoding cDNAs (53).
The present inventors have identified novel mammalian proteins containing both EH and SH3 domains, which have been named Ese1 and Ese2. Sequence and functional analysis of the full length proteins have implicated these proteins in receptor mediated endocytosis via clathrin coated pits and therefore the proteins have been named Ese1 and Ese2 respectively (Ese: for EH-domain and SH3 domain regulator of Endocytosis). Also identified are several mammalian alternative transcript proteins two of which are named Ese1L and Ese2L.